Expression of eNOS and NO production in aortas. (a) Immunoblot analysis for eNOS in aortas. Expression of eNOS in aortas from eNOS-Tg and apoE-KO/eNOS-Tg mice was significantly higher than that in WT mice. Note that the protein levels in aortas from apoE-KO mice or apoE-KO/eNOS-Tg mice were not reduced compared with WT mice or eNOS-Tg mice, respectively, but rather increased in the early phase of atherosclerosis. (b) Immunoblots were analyzed and quantified by densitometry. n = 6 for each group. *P < 0.001 vs. WT mice; **P < 0.05 vs. WT mice; ***P < 0.01 vs. apoE-KO mice. (c–g) NO production in aortas was evaluated by fluorescence detection with DAF-2 DA as described in Methods. (c–f) Shown are representative photographs of the detected NO production in aortas from apoE-KO (c and e) and apoE-KO/eNOS-Tg mice (d and f) before (c and d) and after (e and f) incubation with acetylcholine (1 μmol/l). NO production was clearly visualized by administration of acetylcholine (e and f). (g) Quantitative analysis showed that endothelium-derived NO production in apoE-KO/eNOS-Tg mice was significantly higher than that in apoE-KO mice. Acetylcholine-stimulated NO production was expressed as picowatts per centimeter square. n = 6 for each group. *P < 0.05 vs. apoE-KO mice.